Methods of using PAF-antagonistic diazepines

ABSTRACT

The invention discloses a method of treating an individual having a disorder responsive to PAF-antagonist activity by treating the individual with a compound of formula I or II ##STR1## wherein A is an anellated and optionally substituted benzene or 5- or 6-membered heterocyclic ring; R 5  and Z are each independently hydrogen or an optionally substituted C 1  -C 8  alkyl, alkenyl or alkynyl; R 6  is an optionally substituted phenyl, or is thienyl or is α-pyridyl; Y is CO, CS or CH 2  ; and nontoxic, pharmaceutically acceptable acid addition salts thereof.

The invention relates to pharmaceutical compositions containingdiazepines and having a PAF-antagonistic activity. Many of thediazepines are already known, while others are new.

PAF (platelet activating factor) isacetylglyceryl-ester-phosphorylcholine (AGEPC). This compound is knownas a powerful lipid mediator which is released by animal and humanpro-inflammatory cells. Among these cells are found mainly basophilicand neutrophilic granulocytes, macrophages (from the blood or tissues)and thrombocytes which participate in inflammatory reactions.

In pharmacological experiments, PAF shows the following activities:

(a) bronchoconstriction which is about a hundred times more powerfulthan that of histamine;

(b) a lowering of blood pressure, which is presumably due to directperipheral vasodilation;

(c) triggering of thrombocyte aggregation (demonstrated in vitro and invivo);

(d) pro-inflammatory activity by adhesion and aggregation ofneutrophils, followed by the release of lysosomal enzymes and activationof arachidonic acid metabolism (tested in vitro).

These experimentally demonstrable activities of PAF indicate, directlyor indirectly, possible functions of this mediator in anaphylaxis, inthe pathophysiology of bronchial asthma and in inflammation.

PAF antagonists are needed to clarify further pathophysiologicalfunctions of this mediator in humans and animals and for treatingpathological conditions and diseases in which PAF is involved. Examplesof indications of a PAF antagonist include inflammatory processes of thetracheobronchial tree, acute and chronic bronchitis, bronchial asthma,anaphylactic conditions, allergies and inflammation in the mucousmembranes and skin.

Substances with a PAF-antagonistic activity are already known, forexample substances whose structures resemble that of PAF (EuropeanPatent Application No. 94586; U.S. Pat. No. 4,329,302; Japanese PatentApplications Nos. 57165394, 58035116, 58154512) and11-oxopyridoquinazolines (European Patent Application No. 94080).Moreover, compounds from the following ranges of indications have beeninvestigated for a PAF-antagonistic activity: calcium antagonists,anti-allergic agents, anti-inflammatory agents and α-adrenergic agents.

Surprisingly, it has now been found that numerous substances from agroup of compounds which has hitherto not been taken into consideration,namely the diazepines, have a powerful PAF-antagonistic activity.

In the last twenty years, thousands of molecular variants of diazepineshave been synthesized and tested pharmacologically, biochemically and insome cases clinically as well. The majority of diazepines, particularlythose of the 1,4 series, have anticonvulsive, anxiolytic,muscle-relaxant and, to a greater or lesser extent, sedative activities(S. Garrantini et al. "The Benzodiazepines", Raven Press, New York1973). Of the variety of structures, there are a few exceptions whoseactivity profile shows a different picture. Thus, diazepines which areeffective against bilharzia (Drugs of the future 5, 43 (1980)) andagainst high blood pressure (European Patent Application No. 87850) areknown. Other diazepines were found to have analgesic properties (D.Romer et al. Nature 298, 759 (1982)) and an affinity with the dopaminereceptor (Ruhland and Liepmann, C.I.N.P. Congress, Nuremberg (1983)).The PAF-antagonistic activity of diazepines was never considered.

The invention therefore relates to a method of treating an individualhaving a disorder responsive to PAF-antagonist activity, which methodcomprises treating said individual with one or more compounds offormula: ##STR2## In these formulae A is an anellated ring of formula##STR3## B is an anellated ring of formula ##STR4## R₁ and R₂ are eachindependently hydrogen, a C₁ -C₈ straight-chained or branched alkyl,alkenyl or alkynyl optionally mono- or polysubstituted by halogen,hydroxy, alkoxy, alkylmercapto, amino, alkylamino, dialkylamino,alkylcarbonyl, alkyloxycarbonyl or an acid amide group; cycloalkyl; asaturated carbocyclic or heterocyclic ring condensed on, which maycontain oxygen, sulphur or nitrogen as a heteroatom while thenitrogen-containing ring may carry an alkyl group at the nitrogen atom;halogen, trifluoromethyl, nitro, cyano, optionally substituted amino,alkylmercapto, alkylcarbonyl, alkoxy, alkyloxycarbonyl or an acid amidegroup;

R₃, R₄, R₅ and Z are each independently hydrogen, a C₁ -C₈straight-chained or branched alkyl, alkenyl or alkynyl optionally mono-or polysubstituted by halogen, hydroxy, alkoxy, alkylmercapto, amino,alkylamino, dialkylamino, alkylcarbonyl, alkyloxycarbonyl or an acidamide group;

R₆ is phenyl, which can optionally be substituted, preferably in the 2position, by methyl, methoxy, halogen, nitro or trifluoromethyl, or R₆can be thienyl or α-pyridyl;

Y is <CO, <CS or <CH₂ ; and non-toxic, pharmaceutically acceptable acidaddition salts thereof.

Unless otherwise stated Hal is fluorine, chlorine, bromine or iodine;and alkyl is a C₁ -C₈ straight-chained or branched alkyl. The term "acidamide group" means an aminocarbonyl group which is mono- ordisubstituted by alkyl at the nitrogen; this definition also covers anaminocarbonyl group which is closed, incorporating the nitrogen atom toform a five or six membered ring, while the hetero ring can optionallycontain as a further heteroatom an oxygen, nitrogen or sulphur atom, andany nitrogen atom additionally present in the ring can carry an alkylgroup as substituent.

The above compound of formula I and II are known or can be obtained byknown methods such as those described, for example, in Jeffrey W. H.Watthey et al "Heterocyclic Compounds" Volume 43 (1984), "Azepines" Part2, published by John Wiley & Sons Inc. and L. H. Sternbach "Progress inDrug Research" Volume 22 (1978) page 229-263, Birkhauser Verlag Basel.Compounds of formula I and II which contain a carboxylic acid functionmay be obtained as water-soluble alkali or alkaline earth metal salts.

PHARMACOLOGICAL TEST RESULTS

The PAF-antagonistic activity of compounds of formulae I and II wasinvestigated in terms of the inhibition of blood platelet aggregation invitro and the antagonizing of PAF-induced bronchoconstriction inanesthetized guinea pigs.

1. Inhibition of blood platelet aggregation in vitro

The PAF-induced aggregation of human thrombocytes in vitro is used todetermine PAF-antagonistic activity. To obtain thrombocyte-rich plasma(TRP), blood is taken from an uncongested vein using a plastic syringecontaining 3.8% sodium citrate solution. The ratio of sodium citratesolution to blood is 1:9. After careful mixing the citrate blood iscentrifuged for 20 minutes at 150 g (1200 rpm). The thrombocyteaggregation is measured using the method devised by Born and Cross (G.V. R. Born and M. J. Cross, J. Physiol. 168, 178 (1963)), by adding anaggregation initiator (PAF) to the TRP with constant stirring.

The test substance is added 2 to 3 minutes before aggregation isinitiated in a volume of 10 μl. Either distilled water, ethanol and/ordimethylsulphoxide (each in suitable concentrations) can be used assolvent.

Control mixtures contain corresponding volumes of these solvents. Afterthe initial absorption has been recorded (2 to 3 minutes) aggregation isinduced with PAF (5×10⁻⁸ M).

The maximum of the first aggregation wave is used to assess the effectsof the substance. The PAF-induced maximum absorption rate (=maximumaggregation×100%) is tested at the same time in a parallel mixture(=control mixture in one of the channels of the two-channelaggregometer) as each test mixture (second channel) and is used as a100% value.

The aggregation value attained under the effect of the test substance isgiven as a percentage of the control value, i.e. less than 100% in thecase of an inhibition of aggregation and over 100% in the case of anincrease.

Each test substance is investigated at concentrations of from 10⁻³ to10⁻⁸ M with a random sampling of n=4 with regard to an inhibitory effecton the PAF-induced thrombocyte aggregation. Then aconcentration-activity curve is drawn up using 3 concentrations and theIC₅₀ is determined (concentration giving a 50% inhibition ofaggregation).

2. Antagonism of PAF-induced bronchoconstriction in anesthetized guineapigs

Spontaneously breathing male guinea pigs weighing from 300 to 450 g arepretreated orally with the test substance or a control vehicle 1 hourbefore the intravenous infusion with PAF (30 mg/kg×min). The testanimals are then anesthetized by intraperitoneal route with 2 mg/kg ofurethane, after which the jugular vein, carotid artery and trachea arecannulated. In the control animals the infusion of PAF induces apowerful and long-lasting bronchoconstriction which is measured in termsof the volume of the respiratory tract, compliance and resistance, andalso a lowering of blood pressure. After about 7 to 8 minutes deathoccurs. These effects on respiration and blood pressure and the onset ofdeath are prevented by the PAF antagonists described. Suitble dosagesinclude from about 1 to about 50 mg/kg p.o. and 0.1 to 1.0 mg/kg i.v.

The following Table gives the IC₅₀ of a number of compounds of formulaeI and II:

Compounds of formula:

    __________________________________________________________________________     ##STR5##                                                                                                             PAF-Antag.                            No.    A         Z      R.sub.5                                                                         Y   R.sub.6   IC.sub.50 ; [μMol]                                                                Mp. °C.                 __________________________________________________________________________    1 Clobazam                                                                            ##STR6## CH.sub.3                                                                             H                                                                                ##STR7##                                                                          ##STR8## 250    170-172                        2 Triflubazam                                                                         ##STR9## CH.sub.3                                                                             H                                                                                ##STR10##                                                                         ##STR11##                                                                              200    203-205                                ##STR12##                                                                              H      H                                                                                ##STR13##                                                                         ##STR14##                                                                              470    302-304                        4                                                                                     ##STR15##                                                                              C.sub.2 H.sub.5                                                                      H                                                                                ##STR16##                                                                         ##STR17##                                                                               98    226-227                        5                                                                                     ##STR18##                                                                              CH.sub.3                                                                             H                                                                                ##STR19##                                                                         ##STR20##                                                                              220    222-224                        6                                                                                     ##STR21##                                                                              CH.sub.3                                                                             H                                                                                ##STR22##                                                                         ##STR23##                                                                               66    204-205                        7                                                                                     ##STR24##                                                                              CH.sub.3                                                                             H                                                                                ##STR25##                                                                         ##STR26##                                                                               96    162-164                        8                                                                                     ##STR27##                                                                              CH.sub.3                                                                             H                                                                                ##STR28##                                                                         ##STR29##                                                                              290    220-221                        9                                                                                     ##STR30##                                                                              CH.sub.3                                                                             H                                                                                ##STR31##                                                                         ##STR32##                                                                              >1000  238-239                        10                                                                                    ##STR33##                                                                              CH.sub.3                                                                             H                                                                                ##STR34##                                                                         ##STR35##                                                                              310    217-219                        11                                                                                    ##STR36##                                                                              CH.sub.3                                                                             H                                                                                ##STR37##                                                                         ##STR38##                                                                              210    161-162                        12                                                                                    ##STR39##                                                                              H      CH.sub.3                                                                         ##STR40##                                                                         ##STR41##                                                                              350    316-318                        13                                                                                    ##STR42##                                                                              CH.sub.3                                                                             H                                                                                ##STR43##                                                                         ##STR44##                                                                              >1000  122-124                        14                                                                                    ##STR45##                                                                              (CH.sub.2).sub.2OH                                                                   H                                                                                ##STR46##                                                                         ##STR47##                                                                              285    153-157                        15                                                                                    ##STR48##                                                                              C.sub.2 H.sub.5                                                                      H                                                                                ##STR49##                                                                         ##STR50##                                                                              >100   142-144                        16                                                                                    ##STR51##                                                                              CH.sub.3                                                                             H                                                                                ##STR52##                                                                         ##STR53##                                                                              >50    224-226                        17                                                                                    ##STR54##                                                                              H      H                                                                                ##STR55##                                                                         ##STR56##                                                                              >100                                  __________________________________________________________________________

Compounds of formula: ##STR57##

The compounds of formula I and II can be administered to warm-bloodedanimals topically, orally, parenterally or by inhalation. The compoundsare administered as active ingredients in conventional pharmaceuticalpreparations, e.g. in compositions comprising an inert pharmaceuticalvehicle and an effective dose of the active substance, such as tablets,coated tablets, capsules, lozenges, powders, solutions, suspensions,aerosols for inhalation, ointments, emulsions, syrups, suppositories,etc. The effective dose range of the compounds according to theinvention includes at least 10 to 500, preferably between 25 and 100 mgper dose for oral administration, and between 0.01 and 100, preferablybetween 0.1 and 50 mg per dose for intravenous or intramuscularapplication. Solutions containing 0.01 to 1.0, preferably 0.1 to 0.5% ofactive substance can be used for inhalation.

Some examples of pharmaceutical compositions will now be given in whichcompounds of formula I and II are used as the active ingredient.

Unless otherwise specifically stated, the parts given are parts byweight.

1. Tablets

The tablets include the following ingredients:

    ______________________________________                                        Active substance of formula I and II                                                                0.050 parts                                             Stearic acid          0.010 parts                                             Dextrose              1.890 parts                                             Total                 1.950 parts                                             ______________________________________                                    

Preparation:

The substances are mixed together in known manner and the mixture iscompressed to form tablets, each of which weighs 1.95 g and contains 10to 50 mg of active substance.

2. Ointment

The ointment includes of the following ingredients:

    ______________________________________                                        Active substance of formula I and II                                                                 2.000     parts                                        Sodium pyrosulphite    0.050     parts                                        Mixture (1:1) of cetyl alcohol                                                                       20.000    parts                                        and stearyl alcohol                                                           White Vaseline         5.000     parts                                        Synthetic bergamot oil 0.075     parts                                        Distilled water        ad 100.000                                                                              parts                                        ______________________________________                                    

Preparation:

The ingredients are intimately mixed in known manner to form an ointmentof which 100 g contain 2.0 g of the active substance.

3. Inhalation aerosol

The aerosol includes of the following ingredients:

    ______________________________________                                        Active substance of formula I and II                                                                 1.00     parts                                         Soya lecithin          0.20     parts                                         Propellant gas mixture (Frigen ®)                                                                ad 100.00                                                                              parts                                         (11, 12, and 14)                                                              ______________________________________                                    

Preparation:

The ingredients are mixed together in a manner known per se and themixture is transferred into aerosol containers which contain a meteringvalve releasing between 1 and 20 mg of active substance on eachactuation.

4. Solution for ampoules

The solution includes of the following ingredients:

    ______________________________________                                        Active substance of formula I and II                                                                 5.0      parts                                         Polyethylene glycol    400.0    parts                                         Benzyl alcohol         15.0     parts                                         Ethyl alcohol (95%)    100.0    parts                                         Sodium benzoate        50.0     parts                                         Benzoic acid           1.2      parts                                         Doubly distilled water ad 1000.0                                                                              parts                                         ______________________________________                                    

Preparation:

The active substance is dissolved in benzyl alcohol and thenpolyethylene glycol and alcohol are added. The sodium benzoate andbenzoic acid are dissolved in 250 ml of water, combined with the abovesolution and made up to 1000 ml with water. The resulting solution isfiltered free from pyrogens and the filtrate is transferred underaseptic conditions into 1 ml ampoules which are then sterilized andsealed by fusion. Each ampoule contains 1 to 5 mg of the activesubstance.

5. Suppositories

Each suppository includes:

    ______________________________________                                        Active substance of formula I and II                                                                  1.0     parts                                         Cocoa butter (m.p. 36-37° C.)                                                                  1200.0  parts                                         Carnauba wax            5.0     parts                                         ______________________________________                                    

Preparation:

The cocoa butter and carnauba wax are melted together. At 45° C. theactive substance is added and the mixture is stirred until a completedispersion is formed. The mixture is poured into moulds of a suitablesize and the suppositories are suitably packaged.

We claim:
 1. A method of treating an individual having a disorderresponsive to PAF-antagonist activity, which method comprises treatingsaid individual with a therapeutically effective amount of a compound offormulae ##STR58## wherein A is an anellated ring of formula ##STR59## Bis an anellated ring of formula ##STR60## R₁ and R₂, are eachindependently hydrogen, a C₁ -C₈ straight-chained or branched alkyl,alkenyl or alkynyl optionally mono- or polysubstituted by halogen,hydroxy, alkoxy, alkylmercapto, amino, alkylamino, dialkylamino,alkylcarbonyl, alkyloxycarbonyl or an acid amide group; cycloalkyl; asaturated carbocyclic or heterocyclic ring condensed on, which maycontain oxygen, sulphur or nitrogen as a heteroatom while thenitrogen-containing ring can carry an alkyl group at the nitrogen atom;halogen, trifluoromethyl, nitro, cyano, optionally substituted amino,alkylmercapto, alkylcarbonyl, alkoxy, alkyloxycarbonyl or an acid amidegroup;R₃, R₄ R₅ and Z are each independently hydrogen, a C₁ -C₈straight-chained or branched alkyl, alkenyl or alkynyl optionally mono-or polysubstituted by halogen, hydroxy, alkoxy, alkylmercapto, amino,alkylamino, dialkylamino, alkylcarbonyl, alkyloxycarbonyl, or an acidamide group; R₆ is phenyl, which can optionally be substituted,preferably in the 2 position, by methyl, methoxy, halogen, nitro ortrifluoromethyl, or R₆ can be thienyl or α-pyridyl; Y is <CO, <CS or<CH₂ ; and non-toxic, pharmaceutically acceptable acid addition saltsthereof.
 2. The method of claim 1 which comprises treating withcompounds of the formula ##STR61##
 3. The method of claim 2 wherein Y is<CH₂
 4. The method of claim 3 wherein Y is <CO
 5. The method of claim 4whereinA is ##STR62## Z is C₂ H₅ R₅ is hydrogen; and Y is <CO R₆ is##STR63##
 6. The method of claim 4 which comprises treating withcompounds of the formulaA is ##STR64## Z is CH₃ R₅ is hydrogen; Y is <COR₆ is ##STR65##
 7. The method of claim 4 whereinA is ##STR66## Z is CH₃R₅ is H Y is <CO and R₆ is ##STR67##
 8. A method of claim 4 whereinH is##STR68## Z is CH₃ R₅ is H Y is <CO R₆ is ##STR69##
 9. The method ofclaim 1 which comprises treating with compounds of the formula ##STR70##10. The method of claim 9 whereinA is ##STR71##
 11. The method of claim10 whereinB is ##STR72## R₅ is H R₆ is ##STR73##
 12. B is ##STR74## R₅is H R₆ ##STR75##
 13. The method of claim 10 whereinB is ##STR76## R₅ isH R₆ is ##STR77##